Changes in microbial population and chemical composition of corn stover during field exposure and effects on silage fermentation and in vitro digestibility

Copyright © 2019 by Asian-Australasian Journal of Animal Sciences

This is an open-access article distributed under the terms of the Creative Commons Attribution License

Asian-Australas J Anim Sci Vol. 32, No. 6:815-825 June 2019 https://doi.org/10.5713/ajas.18.0514 pISSN 1011-2367 eISSN 1976-5517

Changes in microbial population and chemical composition of corn stover during field exposure and effects on silage fermentation and in vitro digestibility

Lin Sun^1,2,a, Zhijun Wang^3,a, Ge Gentu^1,*, Yushan Jia¹, Meiling Hou⁴, and Yimin Cai^5,*

Objective: To effectively use corn stover resources as animal feed, the changes in microbial population and chemical composition of corn stover during field exposure, and their silage fermentation and in vitro digestibility were studied.

Methods: Corn cultivars (Jintian, Jinnuo, and Xianyu) stovers from 4 random sections of the field were harvested at the preliminary dough stage of maturity on September 2, 2015. The corn stover exposed in the field for 0, 7, 15, 30, 60, 90, and 180 d, and their silages at 60 d of ensiling were used for the analysis of microbial population, chemical composition, fermentation quality, and in vitro digestibility. Data were analyzed with a completely randomized 3×6 [corn stover cultivar (C)×exposure d (D)] factorial treatment design. Analysis of variance was per­

formed using SAS ver. 9.0 software (SAS Institute Inc., Cary, NC, USA).

Results: Aerobic bacteria were dominant population in fresh corn stover. After ensiling, the lactic acid bacteria (LAB) became the dominant bacteria, while other microbes decreased or dropped below the detection level. The crude protein (CP) and water­soluble carbohydrate (WSC) for fresh stover were 6.74% to 9.51% and 11.75% to 13.21% on a dry matter basis, respectively. After exposure, the CP and WSC contents decreased greatly. Fresh stover had a relatively low dry matter while high WSC content and LAB counts, producing silage of good quality, but the dry stover did not. Silage fermentation inhibited nutrient loss and improved the fermentation quality and in vitro digestibility.

Conclusion: The results confirm that fresh corn stover has good ensiling characteristics and that it can produce silage of good quality.

Keywords: Chemical Composition; Corn Stover; Digestibility; Field Exposure;

Silage Fermentation

INTRODUCTION

Corn (Zea mays L.) is an important crop for food production in China and worldwide. Corn stover includes the leaves, stalks, husks, and cobs, and is often reported as a ratio of dry matter (DM) of corn grain to residue of 1:1. Corn stover is the main crop residue in most of China, with an annual yield of approximately 640 million t/yr [1]. Using corn stover as animal feed has been proven economically viable, not only as a method of disposing of corn stover residue, but also as an alternative livestock feed in regions where corn is the main crop [1]. Corn stover is sometimes incinerated in the field [2], raising concerns about air pollution, fine particle generation, and global warming. Moreover, corn stover is considered an important local resource that can be used by farmers to prepare silage as an animal feed and as a rural fuel [3]. Not only fresh corn stover but also its silage is used as an animal feed; large amounts of dried stover are fed to dairy or beef cattle in some localities, China. However, the dry corn

* Corresponding Authors:

Ge Gentu

Tel: +86-13847171066, Fax: +86-0471-4301371, E-mail: gegentu@163.com

Yimin Cai

Tel: +81-298386365, Fax: +81-298386653, E-mail: cai@affrc.go.jp

1 Key Laboratory of Grassland Resources, Ministry

of Education, P.R. of China, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot 010019, China

2 Inner Mongolia Academy of Agricultural Sciences &

Animal Husbandry, Hohhot, Inner Mongolia, 010030, China

3 Inner Mongolia Institute of Grassland Surveying and Planning, Hohhot, Inner Mongolia 010051, China

4 College of Agriculture, Inner Mongolia University of Nationalities, Tongliao 028000, China

5 Japan International Research Center for Agricultural Science (JIRCAS), Tsukuba, Ibaraki 305-8686, Japan

a These authors contributed equally to this article and are co-first authors.

ORCID Lin Sun

https://orcid.org/0000-0002-0589-2103 Zhijun Wang

https://orcid.org/0000-0003-4470-7289 Ge Gentu

https://orcid.org/0000-0001-9398-718X Yushan Jia

https://orcid.org/0000-0001-7655-9933 Meiling Hou

https://orcid.org/0000-0002-9862-4545 Yimin Cai

https://orcid.org/0000-0003-2650-5210 Submitted Jul 5, 2018; Revised Aug 28, 2018;

Accepted Nov 9, 2018

stover that has been left exposed in the field for a long time is very fibrous, low in nutrients and difficult to digest for animals [1]. Feeding livestock low­quality roughage can result in low production. The major constraint for livestock production in some cold climates, such as Inner Mongolia, is a shortage of good­quality feed, where corn stover or hay is the major source of roughage for livestock in winter. Fresh corn stover has good ensiling characteristics due to its relatively high DM content at harvest, low buffering capacity, and adequate water­soluble carbohydrate (WSC) content [4,5]. Corn stover silage has be­

come the major component of forage for dairy cows under most dietary regimes in China [2,6], and has greater potential to improve ruminant production than the more conventional preserved forages used in corn production areas [7].

Silage preparation and storage are the most effective prepa­

ration techniques for fresh stover [8,9]. Corn stover silage can be beneficial to dairy cattle, beef cattle, or sheep producers because it can provide a locally available feed source at a low cost for animal production [1], and silage is a commonly preserved feed in many countries, including China. The preservation of forage crops as high­quality silage depends on the produc­

tion of enough acids to inhibit the activity of undesirable epiphytic microorganisms under anaerobic conditions, espe­

cially lactic acid bacteria (LAB), which are naturally present on forage crops [10,11].

However, most studies in silage research have analyzed silage fermentation of fresh corn stover, and limited information is available on the effects of field drying corn stover on feed com­

position, silage fermentation, and digestibility. In this study, the changes in the microbial population and chemical com­

position of corn stover during field exposure for up to 180 d and their silage fermentation characteristics were studied. In order to evaluate the nutritional value of the corn stover and silages, we also studied the in vitro digestibility of the stover and silage in a corn production area of Inner Mongolia, China.

MATERIALS AND METHODS

Animal care

Animal experiments were approved by the Committee of Animal Experimentation and were performed under the in­

stitutional guidelines for animal experiments of the College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, China. The experiments were per­

formed according to recommendations proposed by the European Commission (1997) to minimize the suffering of animals.

Corn stover and silage preparation

Local corn (Zea mays L.) cultivars (Jintian, Jinnuo, and Xianyu) were obtained from an experimental field at the Inner Mon­

golia Agricultural University (Huhhot, China). Corn stovers

from 4 random sections of the field were harvested at the pre­

liminary dough stage of maturity on September 2, 2015. Silage was prepared from stover exposed in the field for 0, 7, 15, 30, 60, 90, and 180 d. The ensiling material in triplicate for each cultivar was cut into 20­mm segments with a chopping ma­

chine (TS420; Qufu Machinery Equipment Co., Ltd., Qufu, China), and adequate water added to get a moisture content of approximately 60%. Silage was fermented in laboratory­

scale polyethylene silos (1­L volume, 10­cm diameter, 20­cm length; Shenzhen Guanruilong Chemical Co., Ltd., Shenzhen, China), at approximately 760 g (fresh weight). The silos were sealed with a screw top and plastic tape and stored at ambient temperature (20°C to 25°C). The silos were opened after 60 d of ensiling, and the microbial composition, chemical com­

position, fermentation quality, and in vitro digestibility were analyzed.

Microbiological and chemical analysis

Corn stover and silage samples (10 g) were blended with 90 mL of sterilized water, and serially diluted in sterilized saline solution (8.50 g/L NaCl) from 10^–1 to 10^–5 [12]. The number of LAB was determined using the plate­counting method on MRS agar (Difco Laboratories, Inc., Detroit, MI, USA) incubat­

ed at 30°C for 48 h in an anaerobic box (MGC C­31; Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). Yeasts and molds were counted on potato dextrose agar (Nissui Ltd., Tokyo, Japan) after incubation at 30°C for 96 h, and aerobic bacteria were counted on nutrient agar medium (Qingdao Hope Bio­tech­

nology Co., Ltd., Qingdao, China). All microbial data were transformed to log10 colony­forming units (cfu) based on fresh matter (FM).

The DM contents of corn stover and silage was determined by weighing samples that had been oven­dried at 65°C for 48 h, and the data were corrected for residual moisture at 105°C [13]. The crude ash (CA) content was determined after placing samples in a muffle oven for 3 h at 550°C according to the method of No.942.05 of the AOAC [14]. The organic matter (OM) was calculated as weight loss upon ashing. Total nitro­

gen was measured using a Kjeldahl apparatus (KDY­9830, Shanghai, China) using the procedure of the AOAC [14]. Crude protein (CP) was calculated as follows: total nitrogen×6.25.

Neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined using the ANKOM A200i fiber an­

alyzer (ANKOM Technology, Macedon, NY, USA) [15]. The ether extract (EE) content was determined according to pro­

cedure No.963.15 of the AOAC [14]. The WSC concentration of fresh materials and silages were determined using the method of Kim and Adesogan [16].

For the silage fermentation analysis, 10 g of sample was homogenized with 90 mL of deionized water for 5 min [17].

The extract was filtered through two layers of cheesecloth and a filter paper (8 cm; Aoke Co., Ltd., Taizhou, China), and the

pH was measured using a glass electrode pH meter (STARTER 100/B; OHAUS, Shanghai, China). The ammonia nitrogen (N) content was analyzed using a steam distillation method of the filtrates, and lactic, acetic, propionic, and butyric acids were analyzed by high­performance liquid chromatography, as described by Cai [17].

In vitro incubation and degradability measurements In vitro fermentation was performed in serum bottles follow­

ing the method described by Contreras­Govea et al [18] and Kowalski et al [19]. Rumen fluid was obtained before morn­

ing feeding from four Inner Mongolia semi­fine­wool sheep (wethers) through stomach tube. Animals were fed 40 g of alfalfa hay, 400 g of guinea­grass hay, and 240 g of concentrate containing 10% wheat bran, 25% soybean meal, 65% corn grain, and supplemental vitamins and minerals on a DM basis.

Approximately 1 g ground sample (through 1 mm sieve) was placed in 130­mL serum bottles. Rumen fluid was filtered through four layers of gauze and mixed 1:2 (v/v) to buffer; 60 mL of the mixture was transferred into each serum bottle. The buffer was prepared by the method described by Menke [20]

and consisted of (added in order) 400 mL H2O, 0.1 mL solution A (13.2 g CaCl2 ∙2H2O, 10.0 g MnCl2∙4H2O, 1.0 g CoCl2∙6H2O, 8.0 g FeCl3∙6H2O and made up to 100 mL with H2O), 200 mL solution B (39 g NaHCO3/L, H2O), 200 mL solution C (5.7 g Na2HPO4, 6.2 g KH2PO4, 0.6 g MgSO4∙7H2O and made up to 1,000 mL with H2O), 1 mL resazurine (0.1%, w/v) and 40 mL reduction solution (95 mL H2O, 4 mL l N NaOH and 625 mg Na2S∙9H2O). Each serum bottle was kept under CO2 in a water bath at 39°C, after being capped with a butyl rubber stopper and sealed with an aluminum crimp. In the experiment, the gas production (GP) was released and measured during in vitro incubation at every 4 hours’ point intervals. The cumulative 48 h GP data was the total amount of gas produced every 4 hours. Immediately after 48 h incubation, the undigested solids were precipitated by centrifugation at 1,000×g for 10 min at room temperature and dried in an aerated oven at 65°C for 48 h, and were then assayed for DM and CA. The in vitro de­

gradability of OM (OM­D) was calculated as change in its respective weight. All corn stover and their silages were per­

formed in two separate in vitro experimental runs, and each run consisted of 132 bottles: three cultivars×seven exposure d×three replicates×2 (corn stover+its silage), plus six blanks.

The blanks were the same bottle with no materials. The cumu­

lative 48 h GP was corrected by subtracting GP from blank bottles.

Ruminal pH was measured immediately after collection using a digital pH meter (STARTER 100/B; OHAUS, China), and a 20­mL sample was preserved with 0.5 mL of 9 M sulfuric acid and stored at –20°C for subsequent analysis of total volatile fatty acids (VFAs). The total VFA concentration was measured using an automated gas chromatograph [21].

Statistical analysis

Data on the microorganism population, chemical composi­

tion, and fermentation quality after 60 d of ensiling and in vitro digestibility were analyzed with a completely randomized 3×6 (corn stover cultivar [C]×exposure d [D]) factorial treat­

ment design. Analysis of variance was performed using SAS ver. 9.0 software (SAS Institute Inc., Cary, NC, USA), and the statistical model is as follows:

Yijk = μ+αi+βj+αβij+εijk

Where Yijk is the observation, μ is the overall mean, αi is the effect of corn stover cultivar (i = Jintian, Jinnuo, and Xianyu), βj is the effect of exposure d (j = 0, 7, 15, 30, 60, 90, and 180), αβij is the effect of corn stover cultivar×exposure d, and εijk is the error. The mean values were compared using Duncan’s test [22].

RESULTS

Table 1 presents the chemical composition of corn stover dur­

ing the field exposure. At 0 d of exposure, the three cultivars had similar DM contents (30.21% to 30.67%), which increased by 4% to 25% after 7 to 180 d of exposure. The CP contents of the three cultivars were 6.74% to 9.51% of DM. After 180 d of exposure, the CP content decreased to 3.28% to 4.41% of DM. The NDF and ADF contents in the three cultivars were 43% to 61% and 27% to 38% of DM, respectively. The expo­

sure d means in the three cultivars were similar at 7 and 30 d, and differed significantly (p<0.05) for the other exposure d.

With the increase of exposure time, the WSC content decreased markedly, and the mean WSC in the three cultivars decreased sequentially with increasing exposure time (p<0.05). C, D, and C×D influenced (p<0.0001) the DM, OM, CP, NDF, ADF, and WSC contents. C and D influenced (p<0.0001, p = 0.0006) the EE content, but C×D did not (p = 0.999).

Table 2 presents the microbial population of corn stover over the course of the field exposure. Fresh (0 d of exposure) Jinnuo, Jintian, and Xianyu corn stover contained 10⁴ to 10⁸ cfu/g of FM of LAB, aerobic bacteria, yeasts, and molds. From 7 to 180 d of exposure, these microorganism counts of the three cultivars were 10⁴ to 10⁷ cfu/g of FM. The mean aerobic bacteria and molds counts of Jinnuo and Jintian at 180 d of exposure were significantly (p<0.05) lower than other expo­

sure days. C, D, and C×D influenced (p<0.0001) the counts of all microorganisms.

Table 3 presents the microbial population of corn stover silage after 60 d of ensiling. The LAB counts in the silages of the three cultivars were 10⁴ to 10⁷ cfu/g FM. The mean LAB counts of the silages of the three cultivars from the first few d of exposure (0, 7, and 15 d) were significantly (p<0.05) higher than those of silages after 30, 60, 90, and 180 d of exposure.

During exposure, the aerobic bacteria and yeasts remained around <10³ to 10⁷ cfu/g of FM, and molds in all silages were below the detection level (<10³ cfu/g of FM). C, D, and C×D influenced (p<0.0001) the counts of LAB, aerobic bacteria, and yeasts in corn stover silage.

Table 4 presents the chemical composition of corn stover silage after 60 d of ensiling. The OM contents of the three cul­

tivar silages after 0 to 180 d of exposure were similar, ranging from 93% to 95% of DM. With the increase of exposure time, the CP contents of all corn stover silages significantly (p<0.05) decreased. The highest (p<0.05) and lowest (p<0.05) CP con­

tents in the three cultivars were obtained at the initial (0 d) and final (180 d) exposure times. During exposure, the mean NDF and ADF contents in the silages of the three cultivars were 46%

Table 1. Chemical composition of corn stover during field exposure

Exposure (d) DM (%) OM CP EE NDF ADF WSC

---% of DM--- Jinnuo

0 30.21^f 93.36^c 7.99^a 2.34^a 59.06^a 34.70^b 13.04^a

7 36.42^e 94.31^b 7.64^b 2.23^a 53.29^c 27.44^g 12.84^b

15 47.46^b 93.99^b 7.43^bc 2.11^a 45.46^f 27.74^f 12.21^c

30 55.73^a 96.02^a 7.22^cd 1.98^a 43.54^g 29.17^e 12.32^c

60 42.41^d 94.20^b 7.09^ed 1.94^a 58.02^b 33.53^c 10.87^d

90 47.73^b 93.91^b 6.85^e 1.90^a 49.72^e 33.05^d 9.57^e

180 44.61^c 93.87^b 4.41^f 1.87^a 50.42^d 35.21^a 7.13^f

Jintian

0 30.67^g 94.46^d 9.51^a 2.16^a 45.59^f 30.50^c 13.21^a

7 34.46^f 95.31^b 8.28^b 2.04^ab 46.00^ef 32.08^b 13.05^b

15 35.25^e 96.77^a 8.18^b 1.90^ab 46.32^e 29.09^d 12.78^c

30 46.73^a 95.14^bc 7.94^c 1.79^ab 51.66^c 26.26^e 12.24^d

60 39.42^d 94.91^c 7.13^d 1.75^ab 49.63^d 34.64^a 10.44^e

90 45.54^b 95.37^b 6.33^e 1.65^ab 52.50^b 29.19^d 9.43^f

180 43.68^c 94.58^d 3.28^f 1.60^b 54.65^a 34.74^a 7.21^g

Xianyu

0 30.43^e 94.68^e 6.74^a 1.55^a 60.69^a 34.80^c 11.75^a

7 44.78^d 95.83^b 6.28^b 1.46^ab 51.99^f 31.55^e 11.18^b

15 44.62^e 95.19^d 5.58^c 1.30^ab 54.57^d 29.86^f 10.52^c

30 54.45^a 96.21^a 5.40^c 1.23^abc 56.15^b 35.88^b 10.10^d

60 48.52^c 94.71^e 4.59^d 1.18^abc 54.86^cd 36.15^b 8.82^e

90 49.74^b 95.49^c 4.43^d 1.12^bc 53.63^e 32.97^d 7.47^f

180 48.42^c 95.38^cd 3.63^e 0.85^c 55.21^c 37.83^a 6.30^g

SEM 0.12 0.08 0.16 0.16 0.11 0.11 0.04

Cultivar (C) means

Jinnuo 43.51^b 94.24^c 6.95^b 2.05^a 51.36^b 31.55^b 11.14^b

Jintian 39.39^c 95.22^a 7.24^a 1.84^b 49.48^c 30.93^c 11.19^a

Xianyu 45.85^a 94.95^b 5.24^c 1.24^c 55.30^a 34.15^a 9.45^c

Exposure (d, D) means

0 30.44^g 93.21^e 8.08^a 2.02^a 55.11^a 33.33^c 12.67^a

7 38.55^f 95.15^b 7.40^b 1.91^ab 50.43^e 30.36^e 12.36^b

15 42.44^e 95.32^b 7.06^c 1.77^abc 48.78^f 28.9^f 11.84^c

30 52.30^a 95.79^a 6.85^d 1.67^bcd 50.45^e 30.44^e 11.55^d

60 43.45^d 94.61^d 6.27^e 1.62^cd 54.17^b 34.77^b 10.04^e

90 47.67^b 94.93^c 5.87^f 1.56^cd 51.95^d 31.74^d 8.82^f

180 45.57^c 94.61^d 3.77^g 1.44^d 53.43^c 35.93^a 6.88^g

Significance of main effects and interaction

C < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

D < 0.0001 < 0.0001 < 0.0001 0.0006 < 0.0001 < 0.0001 < 0.0001

C × D < 0.0001 < 0.0001 < 0.0001 0.9999 < 0.0001 < 0.0001 < 0.0001

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; SEM, stand- ard error of the mean.

a-g Means within a column with different superscripts differ (p < 0.05).

to 51% and 26% to 33% of DM, respectively; the EE content decreased gradually (p<0.05). With the increase of exposure time, the WSC content of the silages of the three cultivars, as well as the mean decreased (p<0.05). The C, D, and C×D in­

fluenced (p<0.0001 or p = 0.0017) the OM, CP, EE, NDF, ADF, and WSC contents. C and D influenced (p<0.0001, p = 0.0017,

respectively) EE, but C×D did not (p = 1.0000).

Table 5 presents the fermentation quality of the corn stover silages after 60 d of ensiling. The silages of the three cultivars from corn stover exposed for 0 and 7 d were well preserved, with lower (p<0.05) pH and ammonia­N contents and higher (p<0.05) lactic acid content than those of the silages from stover exposed for 15 to 180 d. The silages prepared from corn stover Table 2. Microbial population of corn stover during field exposure

Exposure (d) LAB Aerobic bacteria Yeasts Molds --- log10 cfu/g of FM --- Jinnuo

0 5.25^a 6.81^cb 5.76^c 5.83^a

7 5.15^ab 7.03^b 5.63^cd 5.42^bc

15 5.03^b 7.56^a 5.45^d 5.35^c

30 4.85^c 6.73^c 5.46^d 5.21^c

60 4.76^cd 6.45^d 5.23^e 5.78^a

90 4.31^e 5.31^e 6.23^b 5.61^ab

180 4.62^d 4.56^f 6.72^a 4.57^d

Jintian

0 5.13^b 7.21^b 5.41^b 5.62^a

7 4.82^c 6.83^c 5.67^a 5.26^b

15 4.53^d 7.57^a 3.58^e 5.12^c

30 4.42^d 6.58^d 4.52^c 5.05^c

60 5.24^b 5.73^e 5.72^a 5.62^a

90 4.52^d 5.72^e 4.13^d 5.37^b

180 5.41^a 2.65^f 3.52^e 4.57^d

Xianyu

0 4.52^b 8.84^a 4.67^c 5.59^a

7 4.31^c 6.43^d 4.31^d 5.31^b

15 5.21^a 6.73^c 5.23^b 5.28^b

30 5.16^a 7.47^b 3.52^e 5.27^b

60 3.65^d 6.35^d 5.32^b 5.57^a

90 3.37^e 5.42^e 5.93^a 5.28^b

180 3.42^e 6.42^d 5.73^a 5.14^b

SEM 0.05 0.07 0.07 0.06

Cultivar (C) means

Jinnuo 4.85^a 6.35^b 5.78^a 5.40^a

Jintian 4.87^a 6.04^c 4.65^c 5.23^b

Xianyu 4.23^b 6.81^a 4.96^b 5.35^a

Exposure (d, D) means

0 4.97^a 7.62^a 5.28^bc 5.68^a

7 4.76^b 6.76^d 5.20^c 5.33^bc

15 4.92^a 7.29^b 4.75^d 5.25^cd

30 4.81^b 6.93^c 4.50^e 5.18^d

60 4.55^c 6.18^e 5.42^a 5.66^a

90 4.07^d 5.48^f 5.43^a 5.42^b

180 4.48^c 4.54^g 5.32^ab 4.76^e

Significance of main effects and interaction

C < 0.0001 < 0.0001 < 0.0001 < 0.0001 D < 0.0001 < 0.0001 < 0.0001 < 0.0001 C × D < 0.0001 < 0.0001 < 0.0001 < 0.0001 The samples stored for 60 d and 180 d was soaked by rain before sampling.

LAB, lactic acid bacteria; cfu, colony-forming unit; FM, fresh matter; SEM, standard error of the mean.

a-g Means within a column with different superscripts differ (p < 0.05).

Table 3. Microbial population of corn stover silages at 60 d of ensiling Exposure (d) LAB Aerobic bacteria Yeasts Molds

---log10 cfu/g of FM--- Jinnuo

0 7.26^b < 10³ 4.81^c ND

7 7.86^a < 10³ 4.53^d ND

15 7.21^b < 10³ 3.13^e ND

30 7.02^c 3.74^b 4.82^c ND

60 6.73^d 5.64^a 4.38^d ND

90 6.85^d < 10³ 7.10^b ND

180 6.75^d < 10³ 7.83^a ND

Jintian

0 7.16^b 7.68^a 3.47^d ND

7 7.42^a 6.47^b 4.21^b ND

15 6.52^d 6.35^b < 10³ ND

30 6.41^ed < 10³ 3.82^c ND

60 6.31^e 3.16^d 4.46^a ND

90 6.50^d 4.73^c 4.23^b ND

180 6.73^c < 10³ < 10³ ND

Xianyu

0 6.52^b 7.68^a 3.82^e ND

7 7.46^a 3.15^f 6.20^b ND

15 7.53^a 5.36^b 4.56^d ND

30 6.02^c < 10³ < 10³ ND

60 5.28^d 3.36^e 6.78^a ND

90 4.46^e 3.78^d 5.41^c ND

180 4.32^e 4.65^c 5.38^c ND

SEM 0.09 0.09 0.05 -

Cultivar (C) means

Jinnuo 7.10^a < 10³ 5.23^a -

Jintian 6.72^b 4.63^a 3.58^c -

Xianyu 5.94^c 4.41^b 4.88^b -

Exposure (d, D) means

0 6.98^b 5.79^a 4.03^d -

7 7.58^a 3.59^d 4.98^c -

15 7.09^b 4.36^b 3.39^f -

30 6.48^c 3.09^f 3.55^e -

60 6.11^d 4.05^c 5.21^b -

90 5.94^e 3.23^e 5.58^a -

180 5.93^e < 10³ 5.19^b -

Significance of main effects and interaction

C < 0.0001 < 0.0001 < 0.0001 -

D < 0.0001 < 0.0001 < 0.0001 -

C × D < 0.0001 < 0.0001 < 0.0001 - LAB, lactic acid bacteria; cfu, colony-forming unit; FM, fresh matter; ND, not detec- tion; SEM, standard error of the mean.

a-e Means within a column with different superscripts differ (p < 0.05).

Table 4. Chemical composition of corn stover silages at 60 d of ensiling

Exposure (d) DM OM CP EE NDF ADF WSC

--- % DM --- Jinnuo

0 33.43^a 94.11^bc 7.89^a 2.36^a 54.62^b 31.63^b 5.87^a

7 33.89^a 93.97^c 7.66^b 2.21^a 50.31^c 24.75^g 5.21^b

15 33.32^a 93.92^c 7.61^b 2.13^a 43.57^f 25.21^f 5.12^b

30 33.48^a 94.46^b 7.55^b 2.01^a 40.62^g 26.83^e 5.15^b

60 33.74^a 95.23^a 7.02^c 1.96^a 55.48^a 30.42^c 4.65^c

90 33.27^a 94.53^b 7.04^c 1.93^a 46.72^e 30.02^d 4.13^d

180 33.89^a 94.28^bc 4.35^d 1.91^a 47.41^d 32.57^a 4.06^d

Jintian

0 33.75^a 94.39^c 8.58^a 2.15^a 40.32^g 27.63^d 6.43^a

7 33.31^a 94.28^c 8.13^b 2.06^ab 43.64^f 30.02^c 6.21^b

15 33.42^a 95.57^a 7.83^c 1.92^ab 45.13^e 26.35^e 6.14^b

30 33.35^a 95.74^a 7.66^c 1.81^b 48.47^c 23.41^f 5.79^c

60 33.74^a 94.83^b 7.46^d 1.76^ab 46.78^d 31.68^b 5.65^d

90 33.21^a 94.91^b 6.72^e 1.68^ab 50.24^b 27.48^d 5.07^e

180 33.85^a 94.57^bc 3.74^f 1.59^b 51.69^a 32.31^a 4.43^f

Xianyu

0 33.00^a 93.25^e 6.10^a 1.56^a 54.68^a 32.52^c 5.15^a

7 33.65^a 93.53^ed 5.88^a 1.42^a 47.52^f 29.46^d 5.07^a

15 33.53^a 94.36^c 5.61^b 1.28^ab 50.68^e 26.73^e 4.32^b

30 33.63^a 95.28^b 5.30^c 1.22^b 53.76^b 32.75^c 4.12^c

60 33.52^a 95.95^a 5.27^c 1.17^ab 52.94^c 33.82^b 4.11^c

90 33.21^a 94.37^c 5.17^c 1.15^ab 51.32^d 29.73^d 4.04^c

180 33.68^a 93.59^d 3.58^d 0.89^b 54.51^a 35.25^a 4.02^c

SEM 0.35 0.07 0.14 0.16 0.1 0.12 0.04

Cultivar(C) means

Jinnuo 33.57^a 94.36^b 7.02^b 2.07^a 48.39^b 28.78^b 4.88^b

Jintian 33.52^a 94.90^a 7.16^a 1.85^b 46.61^c 28.41^c 5.67^a

Xianyu 33.46^a 94.33^b 5.27^c 1.24^c 52.20^a 31.47^a 4.40^c

Exposure (d, D) means

0 33.39^a 93.92^d 7.52^a 2.02^a 49.87^c 30.59^c 5.82^a

7 33.62^a 93.93^d 7.22^b 1.90^ab 47.16^f 28.08^e 5.50^b

15 33.42^a 94.62^b 7.02^c 1.78^abc 46.46^g 26.10^g 5.19^c

30 33.49^a 95.16^a 6.84^d 1.68^bcd 47.62^e 27.66^f 5.02^d

60 33.67^a 95.342^a 6.58^e 1.63^bcd 51.73^a 31.97^b 4.80^e

90 33.23^a 94.60^b 6.31^f 1.59^cd 49.43^d 29.08^d 4.41^f

180 33.81^a 94.15^c 3.89^g 1.46^d 51.20^b 33.38^a 4.17^g

Significance of main effects and interaction

C 0.8274 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

D 0.483 <0.0001 <0.0001 0.0017 <0.0001 <0.0001 <0.0001

C×D 0.9683 <0.0001 <0.0001 0.9999 <0.0001 <0.0001 <0.0001

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; SEM, stand- ard error of the mean.

a-g Means within a column with different superscripts differ (p<0.05).

exposed for 180 d yielded the worst fermentation quality (p<

0.05). Jintian stover had a significantly higher mean of lactic acid content (p<0.05) and lower mean ammonia­N content (p<0.05) than the silage from the other two cultivars. The C, D, and C×D influenced (p<0.0001) the pH and contents of lactic acid, acetic acid, propionic acid, and ammonia­N. The C did not influence (p = 0.2579) the butyric acid content of corn stover silage, but the D (p = 0.025) and C×D did (p = 0.0038).

Table 6 presents the ruminal pH, GP, total VFA content, and OM­D of corn stover and silage. The ruminal pH of the three cultivars at different exposure times were similar (6.12 to 6.30). With the increase of exposure time, the GP, total VFA content, and OM­D of the three cultivars decreased. The high­

est (p<0.05) and lowest (p<0.05) GP, total VFA content, and OM­D in the three cultivars were observed in stover on the initial (0 d) and final (180 d) d of exposure, respectively. The Table 5. Fermentation quality of corn stover silages at 60 d of ensiling

Exposure (d) pH Lactic acid

(% of FM) Acetic acid

(% of FM) Propionic acid (%

of FM) Butyric acid

(% of FM) Ammonia-N (g/kg of FM) Jinnuo

0 3.76^d 1.83^a 0.58^c 0.08^c ND 0.60^f

7 3.90^d 1.07^bc 0.30^d 0.07^c ND 0.55^f

15 4.29^c 0.89^c 0.97^b 0.07^c 0.02^a 1.42^e

30 4.38^bc 1.79^a 0.98^b 0.11^c 0.02^a 3.56^a

60 4.47^b 1.11^bc 1.50^a 0.34^b 0.01^ab 2.95^c

90 4.35^bc 1.23^b 0.81^b 0.15^c ND 2.52^d

180 6.63^a 0.94^c 0.42^cd 0.46^a 0.01^ab 3.37^b

Jintian

0 3.84^d 2.53^a 0.42^c 0.09^d 0.01^ab 0.37^d

7 4.07^c 2.43^a 0.86^b 0.11^d 0.02^a 0.42^d

15 4.25^b 0.83^d 0.42^c 0.37^b 0.02^a 0.67^c

30 4.35^b 0.58^e 0.53^c 0.20^c ND 1.41^b

60 4.28^b 1.32^c 1.17^a 0.53^a 0.01^ab 1.30^b

90 4.32^b 1.52^b 1.20^a 0.09^d 0.01^ab 2.33^a

180 5.64^a 0.85^d 0.41^c 0.51^a ND 2.40^a

Xianyu

0 4.07^c 1.06^b 0.68^bc ND ND 0.67^e

7 4.04^c 1.36^a 0.64^bc ND ND 0.62^e

15 4.28^b 0.89^cd 0.55^c 0.07^cd 0.01^ab 1.52^d

30 4.43^b 0.79^de 0.74^ab 0.13^bc ND 2.43^c

60 4.36^b 0.64^f 0.87^a 0.30^a 0.01^ab 2.65^b

90 4.39^b 0.95^c 0.56^c 0.06^cd ND 2.70^b

180 6.31^a 0.73^ef 0.61^bc 0.20^b 0.02^a 3.25^a

SEM 0.05 0.05 0.05 0.03 0.0049 0.06

Cultivar (C) means

Jinnuo 4.54^a 1.27^b 0.79^a 0.18^b 0.01^a 2.14^a

Jintian 4.39^b 1.44^a 0.72^b 0.27^a 0.01^a 1.27^c

Xianyu 4.55^a 0.92^c 0.66^b 0.11^c 0.01^a 1.98^a

Exposure (d, D) means

0 3.89^e 1.81^a 0.56^ed 0.06^d 0.00^b 0.55^e

7 4.00^d 1.62^b 0.6^d 0.06^d 0.01^b 0.53^e

15 4.27^c 0.87^e 0.65^d 0.17^b 0.02^a 1.20^d

30 4.39^b 1.05^d 0.75^c 0.15^bc 0.01^b 2.47^b

60 4.37^b 1.02^d 1.18^a 0.39^a 0.01^ab 2.30^c

90 4.35^bc 1.23^c 0.86^b 0.10^cd 0.00^b 2.52^b

180 6.19^a 0.84^e 0.48^e 0.39^a 0.01^ab 3.01^a

Significance of main effects and interaction

C < 0.0001 < 0.0001 0.0002 < 0.0001 0.2579 < 0.0001

D < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0247 < 0.0001

C × D < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0038 < 0.0001

FM, fresh matter; ND, not detection; SEM, standard error of the mean.

a-e Means within a column with different superscripts differ (p < 0.05).